This is the second revision of our continuation application. Our goal has been to understand the structure and mechanism of collagen molecule assembly and fibril formation. Type I collagen is a heterotrimer normally composed of two al and one a2 chains. Molecular assembly of type I collagen begins with the registration of the C-propeptides. Efficient procollagen I heterotrimer assembly appears to require that a mechanism for correct chain recognition must exist within the ER. An open question in the biosynthetic pathway remains as to how the different gene products are selected; aligned and subsequently folded into the triple helix. We propose the following three studies. (1) Determine the structures of the C-propeptides and examine in vitro the interactions between the various domains. The different domains of the C-propeptides will be synthesized as GST fusion proteins. The fusion proteins will be crystallized and the chimeric protein structures will be determined using the molecular replacement method. The folding of the comparable C- propeptide domains will be compared. The GST fusion propeptides will also be used to study protein-protein interactions using light-scattering and analytical ultra-centrifugation procedures. (2) Determine the interactions of the C-propeptides under in vivo conditions using the yeast two-hybrid system. The different domains of the C-propeptides will be expressed as fusion proteins with the Gal4 activator domain. These will be tested for interaction with the full length C-propeptides expressed as fusion proteins with the Gal4 DNA binding domain. (3) Identify the cell proteins that bind to C-pro a1 and C-pro a2 using affinity chromatography and the yeast two-hybrid system. The C- propeptide-Gal4 DNA binding region fusion protein will be used as a bait to isolate interacting cell proteins. The proteins isolated will be examined for their role in the C-propeptide interactions. The final study (4) will examine N-telopeptide structures as related to molecular assembly into fibrils and the cross-link patterns that modulate packing. These studies will help in understanding dysfunction in humans.